The in vivo enzyme properties attributable to their intracellular activity and concentration are important determinants of the efficiencies of metabolic pathways. It is well known that many enzymes are able to catalyze very specific chemical reactions with surprising accuracy and efficiency. These enzymes, each catalyzing different but a series of chemical reactions, often cooperate to act and minimize the unnecessary accumulation of metabolic intermediates, and thus form highly integrated metabolic pathways. It is thought that the evolution of enzymes and metabolic pathways are driven in large part by the recruitment of enzymes from other metabolic pathways; enzymes with promiscuous function initially shared by a few distinctive pathways may divergently and cooperatively evolve through gene duplications and subsequent functional specialization depending on the importance of each metabolite, resulting in a mosaic or patchwork of homologous enzymes in two distinct pathways8. Since natural evolution is known to be a highly accomplished designer for in vivo enzyme properties and the efficiencies of metabolic pathways, understanding the mechanisms for molecular evolution might allow for the development of a methodology to redesign efficiencies of constructed synthetic metabolic pathways.
In molecular evolution, the fixation probability of mutations is simply determined by their fitness effects: deleterious (opposed by purifying selection and likely discarded from a population), neutral or nearly neutral (genetic drift), or advantageous (supposed by positive selection and likely fixed to a population)9. However, detailed mechanisms for the molecular basis of adaptations of enzymes and pathways are still largely unclear, as the fitness effects are highly dependent on genotypic and/or phenotypic backgrounds of host organisms. Additionally, impacted by changes in the environment, the fitness effects could also vary even in a population in the same environment due to biological noise10,11. Since it is assumed that the large diversity in protein sequences with orthologous relations are created based on the contributions of mutations to fitness effects, it is thought that changes that are kept to a minimum during the course of evolution may be very essential to maintain in vivo enzyme functions.
Directed evolution, modifying a parent protein such that the modified protein exhibits a desirable property, can be achieved by mutagenizing one or more parent proteins and screening the mutants to identify those having a desired property. A variety of directed evolution methods are currently available for generating protein variants that exhibit altered function, compared to a parent polypeptide. However, currently available methods involve generation of tens of thousands to a million or more mutants, which must be screened to find a few critical mutations. Thus, application of currently available methods is limited by inefficiency of screening the enormous number of mutants that are generated.
There is a need in the art for efficient methods of designing and generating protein variants that exhibit altered properties, without the need for generating and screening large numbers of variants.